A number of therapeutically active quinones are postulated to undergo activation to more reactive metabolites in vivo, by initial reduction to the hydroquinone, rearrangement, and alkylation of macromolecules. In order to assess this theory, two such quinones (mitomycin C and adriamycin) have been examined with physiological reducing agents. We have found that mitomycin C is the more reactive of the two, undergoing reduction by NADH to electrophilic intermediates in a reaction catalysed by several different flavin-dependent enzymes. The electrophilic intermediates are trapped by solvent, by added nucleophiles, and by the protein catalyst itself. Very little of the protein alkylation occurs at the active site, however, but almost exclusively at the exterior surface of the protein. This suggests that the time scale for the rearrangements is greater than that for release of the reduced quinone from the active site. This conclusion implies that for these alkylation processes to have specificity for their target, it is necessary for the quinone to have formed a stable complex with the target macromolecule prior to reduction. The accessibility of these complexes to the enzyme catalyst is being examined.